High efficiency transformation of plants is important in analyzing the usefulness of a variety of genes. Further high efficiency transformation of monocots is also important because large numbers of transgenic plants are needed to study the effect of a particular gene within a given period of time. The ability to directly transform agronomically important plant species at a usable frequency and across a wide range of genetic diversity is important for the development of commercial hybrid products with improved traits including, but not limited to, insect resistance, disease resistance, herbicide resistance, increased yield, increased tolerance to environmental stresses (such as drought, heat, etc.), enhanced seed quality (such as increased or modified starch, oil and/or protein content), and the like.
Genetic improvement of various crop species by genetic engineering has sometimes been hindered because techniques for in vitro culture, transformation, and regeneration of model cultivars are less effective with commercial cultivars. It would be of great benefit to improve the ability to genetically engineer monocots such as maize and sorghum to increase nutritional value, increase resistance to pests, diseases and environmental stress, and to enhance alternative uses.
Additionally, demands for food and fodder are increasing in developing countries in light of growing stress due to population and environment. For example, over the period between 1950 and 1980, the increase in maize production worldwide outpaced both wheat and rice. Despite a temporary downswing in the early to mid-1980's, due to both environmental and political factors, world maize production has risen steadily from around 145 million tons in 1950 to nearly 500 million tons by 1990. Increases in yield and harvested area have been the predominant contributors to enhanced world production; with yield playing the major role in industrialized countries and area expansion being most important in developing countries. Yet, over the next ten years it is also predicted that meeting the demand for corn worldwide will require an additional 20% over current production (Dowswell, C. R., Paliwal, R. L., Cantrell, R. P. (1996) Maize in the Third World, Westview Press, Boulder, Colo.).
Sorghum (Sorghum bicolor (L.) Moench) is a widely grown grain and forage crop, and is more closely related than rice to the major crops of tropical origin such as maize, sugarcane, and pearl millet. Sorghum ranks fifth worldwide in production among cereal crops, and is an important model for tropical grasses of worldwide importance. It is unique among major cereals because it adapts well to environmental extremes, notably drought and heat. These attributes make sorghum the logical grain to support human and animal populations in areas with extreme heat and minimal precipitation. Even in the absence of drought, water availability is an emerging problem that will affect at least six billion people worldwide by 2025. Increased demand for limited fresh water, coupled with global climate trends, and expanding populations, will increase the attractiveness of dry land crops such as sorghum. Moreover, it is second only to maize within the U.S. as a feedstock for ethanol production.
Several laboratories have reported successful but low rates of transformation frequency in sorghum utilizing particle bombardment (Able et al. 2001, In Vitro Cell Dev Biol, 37:341; Casas et al. 1993, Proc Natl Acad Sci., USA 90: 11212) or Agrobacterium-mediated transformation with the bar gene (Zhao et al. 2000, Plant Mol Biol 44:789) or the nptIl gene (Tadesse et al. 2003, Plant Cell Tissue Organ Cult 75:1, Howe et al., 2006 Plant Cell Rep.; 25:784-791) as selectable markers. Recently, an Agrobacterium-based system was coupled with a visual marker gene selection strategy to identify sorghum transformants (Gao Z, Jayaraj J, Muthukrishnan S, Claflin L, Liang G H (2005) Genome 48:321). The microprojectile systems previously reported are hampered by reproducibility and relatively low efficiencies. On the other hand, Agrobacterium-mediated transformation is relatively efficient for sorghum using the ‘super binary’ vector system (Ishida et al. 1996, Nat Biotechnol: 14:745), short subculture intervals, and the addition of PVPP to tissue culture media (Zhao et al. 2000, Plant Mol. Biol 44:789). These latter two steps help block the negative impacts of associated phenolic production from sorghum tissue. Demonstration of standard binary plasmids being suitable for Agrobacterium-mediated transformation of sorghum (Gao et al. 2005 Genome 48:321) and a novel Agrobacterium vector (Howe et al. 2006, Plant Cell Rep.; 25:784-791) are reported.
Even with the advances listed above, transformation frequencies in sorghum are low and reported to average 1% and be as high as 5% (Nguyen et al., 2003, Plant Cell Tiss Organ Cult. DOI 10.1007/s11240-007-9228-1). Visarada and Kishore report that transformation followed by regeneration remains extremely complicated in sorghum transgenic technology. (Infor. Syst. for Biotech. March, 2007; pp. 1-3). There is a need, therefore, for efficient methods for transformation and regeneration that can be used with sorghum as well as a wide variety of monocots.